Biologically active hemagglutinin from type a clostridium botulinum and methods of use

ABSTRACT

An isolated, biologically active 33 kDa hemagglutinin purified from the type A  Clostridium botulinum  neurotoxin complex and its uses are described.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government grants from the Naval ResearchLaboratory/United States Army Edgewood Research, Development, andEngineering Center (N00014-92-K-2007), the U.S. Department ofAgriculture (94-37201-1167), and the National Institutes of Health(NS33740). The Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to the isolation of a biologically activehemagglutinin protein from the Clostridium botulinum type A neurotoxincomplex.

Botulinum neurotoxins (BoNTs) are extremely potent proteins with a mouselethal dose of 0.3 ng/kg. Seven serotypes (A-G) of BoNTs are produced bydifferent strains of Clostridium botulinum. In addition, certainnonbotulinum species of Clostridium, such as C. butyricum and C.baratii, have been shown to produce the toxins. The neurotoxin is aprotein of about 150 kDa and consists of two polypeptide chains (a 50kDa light chain and a 100 kDa heavy chain), which are linked togethervia a disulfide bond.

BoNTs are often produced by bacteria under anaerobic conditions inimproperly stored or processed foods. Ingestion of the contaminated foodcan cause the flaccid muscle paralysis known as botulism.

The mechanism by which BoNTs cause botulism has been well studied. Afteringestion, the neurotoxin is translocated across the intestinal mucosa,gaining access to neuromuscular junctions. At affected junctions, theneurotoxin is internalized by neurons via endocytosis. Inside the cell,the toxin's protease activity degrades specific vesicular and plasmamembrane proteins, disrupting neurotransmitter release from the neuron.Thus, the patient experiences paralysis due to an inability to releaseneurotransmitters from the presynaptic surface. This process is reviewedin Sakaguchi, Pharmac. Ther., 19:165-194 (1983).

SUMMARY OF THE INVENTION

The invention is based on the isolation of biologically active, proteaseresistant hemagglutinin from the type A Clostridium botulinum neurotoxincomplex. This protein is estimated to have a molecular weight of 33 kDaby sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE),and therefore this protein and corresponding polypeptides are referredto herein as “Hn-33.” Hn-33 is “biologically active” if the polypeptideexhibits hemagglutinin activity in a hemagglutination assay (describedbelow).

The isolated, biologically active Hn-33 can be used in oral vaccines toprotect the antigen from degradation in the gastrointestinal (GI) tractor, alternatively, in a pharmaceutical composition suitable forinjection into a muscle or its surrounding tissue to alleviate abnormalfiring of neuromuscular junctions.

In general, the invention features a composition including an isolated,biologically active Hn-33 from type A Clostridium botulinum and at leastone neurotoxin, e.g., type A botulinum neurotoxin, type E botulinumneurotoxin, or tetanus toxin. The composition can further include apharmaceutical carrier suitable for administration to an animal. Thepharmaceutical carrier can be an adjuvant, or a separate adjuvant can beadded. The pharmaceutical carrier can include any one or more of, e.g.,polysorbates, ethanol, starch, and glycerin.

In another aspect, the invention features an isolated, biologicallyactive Hn-33 polypeptide of type A Clostridium botulinum. An isolatedHn-33 polypeptide is one that has been separated from components whichnaturally accompany it. Typically, the protein is isolated when it is atleast 60%, by weight, free from the proteins and naturally-occurringorganic molecules with which it is naturally associated. Preferably, thepreparation is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, Hn-33. Such an isolated Hn-33 can beobtained, for example, by fractionating a type A C. botulinum culture,by expression of a nucleic acid encoding Hn-33, or by chemicallysynthesizing the protein. Purity can be measured by any appropriatemethod, e.g., column chromatography or gel filtration. Biologicallyactive Hn-33 must be isolated in a way to ensure that the resultingpolypeptide maintains hemagglutinin activity.

The Hn-33 polypeptide of the invention need not be a full-lengthpolypeptide nor be identical to the wild-type amino acid sequence. Hn-33polypeptides containing truncations, deletions, or amino acidsubstitutions are included, as long as the polypeptide or protein isbiologically active as described herein. As used herein, the terms“polypeptide” and “protein” are used interchangeably.

The invention also features a method of eliciting an immune responseagainst a neurotoxin, e.g., type A botulinum neurotoxin, type Ebotulinum neurotoxin, or tetanus toxin, in an animal, e.g., a human or awild or domesticated animal such as a dog, cat, bird, cow, pig, orhorse, by administering to the animal, e.g., orally, an amount of acomposition effective to elicit an immune response against theneurotoxin, the composition including an isolated, biologically activeHn-33 of type A Clostridium botulinum and a neurotoxin. The compositioncan further include an adjuvant and/or a biodegradable polymer thatenables slow release of the Hn-33/toxin composition.

In another aspect, the invention features a method of treating orpreventing a neuromuscular condition, e.g., a smooth muscle spasm or askeletal muscle spasm, in an animal, e.g., a human, by administering tothe animal an amount of a composition effective to treat or prevent theneuromuscular condition, the composition including an isolated,biologically active Hn-33 of type A Clostridium botulinum and aneurotoxin, e.g., type A botulinum neurotoxin, type E botulinumneurotoxin, or tetanus toxin. For example, the composition can beinjected into a muscle in need of treatment or a tissue surrounding themuscle. The composition can further include a polymer that enables slowrelease of the neurotoxin. The method also can be used to treat certainneuromuscular headaches and cerebral palsy.

A “neurotoxin” (or “toxin”) is any active, attenuated, or inactivepolypeptide that inhibits neurotransmitter release from a presynapticmembrane of a neuromuscular junction. When used in a vaccinecomposition, the neurotoxin is preferably inactive or at leastattenuated. When used in a therapeutic composition to treat abnormalfiring of a neuromuscular junction, the neurotoxin is preferably fullyactive. Suitable neurotoxins for use in the compositions and methods ofthis invention include, for example, type A botulinum neurotoxin, type Ebotulinum neurotoxin, and tetanus toxin.

An “adjuvant” is a substance that is incorporated into or isadministered simultaneously with the vaccine compositions of theinvention. Adjuvants increase the duration or level of the immuneresponse in the animal after administration of the polypeptide orpeptide mimetic. An adjuvant can also facilitate delivery of thepolypeptide or peptide mimetic into the animal or into specific tissues,cells, or locations throughout the body of the animal. Examples ofadjuvants include, but are not limited to, incomplete Freund's, completeFreund's, and alum; and can contain squalene (e.g., MF59, Chiron Corp,Emeryville, Calif.), monophospholipid A (e.g., Detox™, Ribi ImmunoChemResearch, Inc., Hamilton, Mont.), saponins (QS-21, Cambridge Biotech,Cambridge, Mass.), non-ionic surfactants (NISV, Proteus, Cheshire,United Kingdom), tocols (U.S. Pat. No. 5,667,784),biodegradable-biocompatible poly(D,L-lactide-co-glycolide) (U.S. Pat.No. 5,417,986), immune-stimulating complexes (ISCOMs), and/or liposomes.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although suitable methods andmaterials for the practice or testing of the present invention aredescribed below, other methods and materials similar or equivalent tothose described herein, which are well known in the art, can also beused. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2, 4, and 7 are elution profiles from chromatography columns.The profile in FIG. 1 is a DEAE-Sephadex A-50 column separation of acrude type A C. botulinum extract. The profile in FIG. 2 is a SephadexG-100 column separation of a crude type A botulinum neurotoxin complex.The profiles in FIG. 4 are a Sephadex G-200 column separation ofisolated, biologically active Hn-33 (in black) and standard proteins (ingray). The profile in FIG. 7 is a Sephadex G-200 column separation of anHn-33/type A neurotoxin composition.

FIGS. 3A and 3B are graphs of a matrix assisted laser desorption massspectrum of Hn-33. The spectrum in FIG. 3B extends and enlarges thespectrum in FIG. 3A.

FIG. 5 is a graph of far-UV CD spectra of Hn-33.

FIGS. 6A and 6B are graphs of IR spectra of Hn-33.

FIG. 8 is a bar graph showing cleavage of SNAP-25 substrate by botulinumneurotoxins.

FIG. 9 is the amino acid sequence of the full-length Hn-33 polypeptide.

DETAILED DESCRIPTION

The invention relates to isolated, biologically active, proteaseresistant hemagglutinin polypeptides from the type A Clostridiumbotulinum neurotoxin complex referred to herein as Hn-33. Hn-33 can beused in vaccines, e.g., oral vaccines, to protect the antigen fromdegradation in the GI tract, or in a pharmaceutical composition suitablefor injection into a muscle or its surrounding tissue to alleviateabnormal excitation of a neuromuscular junction.

I. Production and Isolation of Biologically Active Hn-33

A. Production

Biologically active Hn-33-derived proteins and polypeptides include anyprotein or polypeptide sharing a functional characteristic withbiologically active, wild type Hn-33. Such functionally related,biologically active Hn-33 polypeptides include polypeptides havingadditions or substitutions of amino acid residues within the Hn-33 aminoacid sequence compared to the wild type Hn-33 sequence (see FIG. 9 andEast et al., System. Appl. Microbiol. 17:306-312, 1994; SEQ ID NO:1).Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved.

For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

Biologically active Hn-33 variants with altered amino acid sequences canbe created by making random mutations to Hn-33 DNA (which has beendescribed in East et al., Curr. Microbiol., 29:69-77, 1994) using randommutagenesis techniques well known to those skilled in the art, andisolating the polypeptide in a way that yields biologically activeHn-33. Alternatively, site-directed mutations of the Hn-33 codingsequence can be engineered using techniques also well-known to thoseskilled in the art.

To design variant biologically active Hn-33 polypeptides, it is usefulto distinguish between conserved positions and variable positions. Toproduce variants with functions most similar to wild type, biologicallyactive Hn-33, it is preferable that conserved residues are not altered.Moreover, alteration of non-conserved residues are preferablyconservative alterations, e.g., a basic amino acid is replaced by adifferent basic amino acid. To produce altered function variants, it ispreferable to make non-conservative changes at variable and/or conservedpositions. Deletions at conserved and variable positions can also beused to create variants.

Other mutations to the Hn-33 coding sequence can be made to generatebiologically active Hn-33 polypeptides that are better suited forexpression, e.g., for scaled up production, in a selected host cell.

Biologically active Hn-33 polypeptides test positive in ahemagglutination assay (described below), and have a specified function(i.e., protects a therapeutic protein drug from degradation). Indetermining whether a particular biologically active Hn-33 polypeptideor variant thereof is functional, one can use any assay techniquesdisclosed herein or techniques disclosed in references incorporatedherein. Biologically active Hn-33 polypeptides and variants can have40%, 60%, 75%, 95%, 100%, or an even higher percentage, of the activityof the full-length, biologically active, wild type Hn-33 (i.e., avariant can have higher activity than the wild type Hn-33). Suchcomparisons are generally based on equal concentrations of the moleculesbeing compared. The comparison can also be based on the amount ofprotein or polypeptide required to reach 50% of the maximal activityobtainable.

In general, biologically active Hn-33 can be isolated from its naturalhost, type A Clostridium botulinum bacteria (A.T.C.C. Nos. 3502, 17862,19397, 25763, and 51385). Alternatively, biologically active Hn-33polypeptides according to the invention can be produced bytransformation (transfection, transduction, or infection) of a host cellwith an Hn-33-encoding DNA fragment in a suitable expression vehicle.Suitable expression vehicles include plasmids, viral particles, andphage. For insect cells, baculovirus expression vectors are suitable.The entire expression vehicle, or a part thereof, can be integrated intothe host cell genome. In some circumstances, it is desirable to employan inducible expression vector, e.g., the LACSWITCH™ InducibleExpression System (Stratagene; LaJolla, Calif.).

Those skilled in the field of molecular biology will understand that anyof a wide variety of expression systems can be used to provide therecombinant protein. The precise host cell used is not critical to theinvention.

Proteins and polypeptides can also be produced by plant cells. For plantcells, viral expression vectors (e.g., cauliflower mosaic virus andtobacco mosaic virus) and plasmid expression vectors (e.g., Ti plasmid)are suitable. Such cells are available from a wide range of sources(e.g., the American Type Culture Collection, Rockland, Md.; also, see,e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, New York, 1994). The methods of transformation or transfectionand the choice of expression vehicle will depend on the host systemselected. Transformation and transfection methods are described, e.g.,in Ausubel et al. (Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1994); expression vehicles may be chosen from thoseprovided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwelset al., 1985, Supp., 1987).

The host cells harboring the expression vehicle can be cultured inconventional nutrient media adapted as need for activation or repressionof a chosen gene, selection of transformants, or amplification of achosen gene.

Specific initiation signals may be required for efficient translation ofinserted nucleic acid sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where an entire nativebiologically active Hn-33 gene, including its own initiation codon andadjacent sequences, is inserted into the appropriate expression vector,no additional translational control signals may be needed. In othercases, exogenous translational control signals, including, perhaps, theATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription or translationenhancer elements, (e.g., ones disclosed in Bittner et al., Methods inEnzymol., 153:516, 1987).

Preferred polypeptides are those which are soluble under normalphysiological conditions. Also within the invention are fusion proteinsin which a portion (e.g., one or more domains) of biologically activeHn-33 is fused to an unrelated protein or polypeptide (i.e., a fusionpartner) to create a fusion protein. The fusion partner can be a moietyselected to facilitate purification, detection, or solubilization, or toprovide some other function. Fusion proteins are generally produced byexpressing a hybrid gene in which a nucleotide sequence encoding all ora portion of biologically active Hn-33 is joined in-frame to anucleotide sequence encoding the fusion partner. For example, theexpression vector pUR278 (Ruther et al., EMBO J., 2:1791, 1983), can beused to create lacZ fusion proteins. The pGEX vectors can be used toexpress foreign polypeptides as fusion proteins containing glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan be easily purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

A fusion protein can be readily purified by utilizing an antibodyspecific for the fusion protein being expressed. For example, a systemdescribed in Janknecht et al., Proc. Natl. Acad. Sci. USA., 88:8972(1981), allows for the ready purification of non-denatured fusionproteins expressed in human cell lines. In this system, the gene ofinterest is subcloned into a vaccinia recombination plasmid such thatthe gene's open reading frame is translationally fused to anamino-terminal tag consisting of six histidine residues. Extracts fromcells infected with recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose columns, and histidine-tagged proteins areselectively eluted with imidazole-containing buffers. The same procedurecan be used for a bacterial culture, e.g., a type A Clostridiumbotulinum culture.

Alternatively, biologically active Hn-33 or a portion thereof, can befused to an immunoglobulin Fc domain. Such a fusion protein can bereadily purified using an affinity column.

Polypeptides of the invention, particularly short, biologically activeHn-33 fragments, can also be produced by chemical synthesis (e.g., bythe methods described in Solid Phase Peptide Synthesis, 2nd ed., [1984]The Pierce Chemical Co., Rockford, Ill.).

B. Isolation

Both naturally occurring and recombinant forms of Hn-33 must be isolatedand tested for biological activity. Secreted forms can be isolated fromthe culture media, while non-secreted forms must be isolated from thehost cells. Polypeptides can be isolated by affinity chromatography. Inone example, an anti-Hn-33 protein antibody (produced as describedherein) is attached to a column and used to isolate the biologicallyactive Hn-33 protein. Lysis and fractionation of biologically activeHn-33-harboring cells prior to affinity chromatography can be performedby standard methods (see, e.g., Ausubel et al., supra). Alternatively, abiologically active Hn-33 fusion protein, for example, a biologicallyactive Hn-33-maltose binding protein, a biologically activeHn-33-β-galactosidase, or a biologically active Hn-33-trpE fusionprotein, can be constructed and used to isolate biologically activeHn-33 (see, e.g., Ausubel et al., supra; New England Biolabs, Beverly,Mass.).

For example, a C. botulinum culture is grown in N-Z amine-based growthmedium and the cells harvested. After acid precipitation andcentrifugation, the cells are lysed and treated with RNAse A. The cellextracts can be fractionated on a DEAE-Sephadex A-50 column, and thepooled fractions precipitated with ammonium sulfate. The precipitatedproteins are then redissolved and fractionated on a G-100 Sephadexcolumn to yield isolated, biologically active Hn-33.

Once isolated, the Hn-33 polypeptide can, if desired, be furtherpurified and/or concentrated, as long as further processing does notimpair biological activity (as measured in the hemagglutination assaydescribed herein). A variety of methods for purification andconcentration are well known in the art (see, e.g., Fisher, LaboratoryTechniques In Biochemistry And Molecular Biology, eds., Work and Burdon,Elsevier 1980), including ultracentrifugation and/or precipitation(e.g., with ammonium sulfate), microfiltration (e.g., via 0.45 μmcellulose acetate filters), ultrafiltration (e.g., with the use of asizing membrane and recirculation filtration), gel filtration (e.g.,columns filled with Sepharose CL-6B, CL-4B, CL-2B, 6B, 4B or 2B,Sephacryl S-400 or S-300, Superose 6 or Ultrogel A2, A4, or A6; allavailable from Pharmacia), fast protein liquid chromatography (FPLC),and high performance liquid chromatography (HPLC).

C. Physical Properties

After isolation procedures have been performed, the concentration andpurity of biologically active Hn-33 can readily be determined. Forexample, the polypeptide concentration and yields can be determined by avariation of the Bradford assay, using a reagent sold by Bio-Rad. Thepurity can be estimated by, for example, the banding resulting fromassaying the protein sample on SDS-PAGE.

Other physical properties of Hn-33 variant proteins can be determined.The molecular weight of the proteins can be estimated by methods wellknow in the art, such as SDS-PAGE, laser desorption mass spectroscopy,and column chromatography. In addition, the secondary structure can beanalyzed by circular dichroism analysis as described in Fu et al., Appl.Spectrosc., 48:1432-1441 (1994).

II. Characterization of Hn-33

A. Biological Activity

Isolated Hn-33 proteins can be assayed for biological activity using thehemagglutination assay as described in DasGupta et al., Can. J.Microbiol., 23:1257-1260 (1977) and Example 2 below. Briefly, washed redblood cells (e.g., 0.05 ml of a 0.5% w/w suspension) are added to asmall volume (e.g., 50 μl solution) of a Hn-33 polypeptide sample (e.g.,1 ng/ml to 15 or 20 μg/ml, e.g., 416 ng/ml) in a U-bottom well of amicrotiter plate. Hemagglutination activity is observed when the redblood cells clump and thus do not form an annular shape or a dot at thebottom of the well within 30 minutes after adding the Hn-33. An Hn-33polypeptide is considered biologically active if a concentration ofHn-33 of as low as 300 ng/ml results in hemagglutination in the aboveassay.

B. Function

An isolated, biologically active Hn-33 polypeptide can be assayed forthe percentage of biologically active, wild-type Hn-33 protein functionas described in Examples 2 and 6 below. Briefly, the Hn-33 andneurotoxin proteins are mixed in a Hn-33:neurotoxin, by weight, ratio ofat least 1. The mixture is dialyzed against a protease digestion bufferfor 30 minutes, digested for at least 20 minutes, and subsequentlyanalyzed by SDS-PAGE and standard visualization procedures (e.g.,Coomassie Blue staining of the gel). The biologically active Hn-33polypeptide preferably exhibits at least 40, 60, 80, 90, 100 percent, oreven a higher percentage, of the protective function of the wild type,biologically active Hn-33 protein.

C. Antibodies

Biologically active Hn-33 polypeptides can be used to generateanti-Hn-33 antibodies, e.g., antibodies that are specific for thebiologically active form of the protein. Such biologicallyactive-specific antibodies can be used in diagnostic and therapeuticprocedures that require the enhancement, inhibition, or detection of thetype A neurotoxin complex.

Biologically active Hn-33 polypeptides (or immunogenic fragments oranalogs) can be used to raise antibodies. In general, the biologicallyactive Hn-33 can be coupled to a carrier protein such as KLH (asdescribed in Ausubel et al., supra), mixed with an adjuvant, andinjected into a host mammal to produce polyclonal antibodies. Theseantibodies can be purified by antigen affinity chromatography.Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of the immunized animals.

In particular, various host animals can be immunized by injection with abiologically active Hn-33 protein. Host animals can include rabbits,mice, guinea pigs, and rats. Various adjuvants can be used to increasethe immunological response, depending on the host species, including butnot limited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

Antibodies include polyclonal antibodies, monoclonal antibodies,humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, and molecules produced using a Fabexpression library.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be prepared using the biologically activeHn-33 polypeptides described above and standard hybridoma technology(see, e.g., Kohler et al., Nature, 256:495, 1975; Kohler et al., Eur. J.Immunol., 6:511, 1976; Kohler et al., Eur. J. Immunol., 6:292, 1976;Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y., 1981; U.S. Pat. No. 4,376,110; Kosbor et al., ImmunologyToday, 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA, 80:2026,1983; Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96, 1983; and Ausubel et al., supra). Such antibodiescan be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD andany subclass thereof. The hybridoma producing the Mab of this inventionmay be cultivated in vitro or in vivo. The ability to produce hightiters of mAbs in vivo makes this the presently preferred method ofproduction.

Once produced, polyclonal or monoclonal antibodies are tested forspecific biologically active Hn-33 recognition by Western blot orimmunoprecipitation analysis by standard methods, e.g., as described inAusubel et al., supra. Antibodies that specifically recognize and bindto biologically active Hn-33 can be used, e.g., in an immunoassay tomonitor the level of biologically active Hn-33 or type A neurotoxinproduced by a Clostridium botulinum-infected mammal (for example, todetermine the amount or subcellular location of biologically activeHn-33).

Antibodies of the invention can be produced using fragments of thebiologically active Hn-33 protein which lie outside highly conservedregions and appear likely to be antigenic, e.g., based on criteria suchas high frequency of charged residues. Such fragments can include adiscontinuous, biologically active-specific epitope as well. In onespecific example, such fragments are generated by standard techniques ofPCR and are then cloned into the pGEX expression vector (Ausubel et al.,supra). Fusion proteins are expressed in E. coli and purified using aglutathione agarose affinity matrix as described in Ausubel, et al.,supra.

In some cases it may be desirable to minimize the potential problems oflow affinity or specificity of antisera. In such circumstances, two orthree separate fusion proteins can be generated, and each fusion can beinjected into at least two rabbits. Antisera can be raised by injectionsin a series, preferably including at least three booster injections.

Antisera is also checked for its ability to immunoprecipitaterecombinant biologically active Hn-33 polypeptides or control proteins,such as glucocorticoid receptor, CAT, or luciferase.

The antibodies can be used, for example, to detect biologically activeHn-33 in a biological sample as part of a diagnostic assay, and also toevaluate the effectiveness of medical treatments by other therapeuticapproaches. Antibodies also can be used in a screening assay to measurethe effect of a candidate compound on localization of biologicallyactive Hn-33.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851, 1984;Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452,1984) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine Mab and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can beadapted to produce single chain antibodies against a biologically activeHn-33. Single chain antibodies are formed by linking the heavy and lightchain fragments of the Fv region via an amino acid bridge, resulting ina single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include, butare not limited to, F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science, 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to the biologically active Hn-33 can, in turn, be used togenerate anti-idiotype antibodies that resemble a portion ofbiologically active Hn-33, using techniques well known to those skilledin the art (see, e.g., Greenspan et al., FASEB J., 7:437, 1993;Nissinoff, J. Immunol., 147:2429 1991). For example, antibodies thatbind to biologically active Hn-33 and competitively inhibit the bindingof a ligand of biologically active Hn-33 (e.g.,o-nitropheny-β-D-galactoside or isopropyl-β-D-thiogalactoside) can beused to generate anti-idiotypes that resemble a ligand binding domain ofbiologically active Hn-33 and, therefore, bind and neutralize a ligandof biologically active Hn-33. Such neutralizing anti-idiotypicantibodies or Fab fragments of such anti-idiotypic antibodies can beused in therapeutic regimens.

D. Protease Resistance

Biologically active Hn-33 or a complex containing biologically activeHn-33 has now been shown to be resistant to a variety of proteases. Thisprotease resistance can be tested by methods well known in the art. Forinstance, a pre-determined amount of Hn-33 or Hn-33 complex (e.g., 10 μgto 100 mg) is mixed with a solution containing a selected protease(e.g., trypsin, chymotrypsin, subtilisin, pepsin, cathepsin B, ancord,asparaginase, bromelain, clostraipain, ficin, kallikarein, or papain) ata pre-determined w/w ratio (e.g., 25:1 to 1000:1 Hn-33:protease ratio)for a pre-determined length of time (e.g., 10 minutes to 24 hours). Thedigestion is carried out at a temperature (e.g., room temperature or 37°C.) and pH (e.g., 2.0 to 8.0) suitable for each protease and can bestopped by adding to the digest any number of protease inhibitors knownin the art (e.g., PMSF).

E. Effect on Neurotoxin Protease Activity

Biologically active Hn-33 can have an effect on the protease activity ofa number of neurotoxins (e.g., botulinum type A neurotoxin, botulinumtype E neurotoxin, and tetanus toxin). The protease activity of aneurotoxin complex containing an isolated, biologically active Hn-33 canbe measured using any suitable substrate (e.g.,glutathione-S-transferase-SNAP-25 fusion protein, VAMP-2, Syntexin—fortype C1 botulinum neurotoxin only, or Cellubrevin) in which at least oneof the cleavage products (e.g., SNAP-25) is detectable (e.g., by Westernblotting using an antibody which binds to the SNAP-25 C-terminus).Additional agents (e.g., sugars or detergents such as SDS) can be addedto the digestion to test the additional agent's effect on neurotoxinprotease activity.

III. Preparation of Pharmaceutical Compositions Containing BiologicallyActive Hn-33

Since biologically active Hn-33 protects botulinum neurotoxin type Afrom proteolytic degradation (shown in Example 6 below), Hn-33 is usefulas a carrier protein in the preparation of pharmaceutical compositionssuch as vaccines or therapeutics.

The isolation and purification of Hn-33 and type A and type E botulinumneurotoxins are described below. Tetanus toxin can be isolated andpurified according to the method described in Schiavo et al., MethodsEnzymol. 248:643-652 (1995).

A. Vaccine Compositions

The invention includes vaccine compositions (e.g., oral vaccines)containing at least one neurotoxin (e.g., type A botulinum neurotoxin,type E botulinum neurotoxin, or tetanus toxin), which can be active orinactive, biologically active Hn-33 (or a biologically activeimmunogenic fragment or derivative thereof), and, optionally, apharmaceutically acceptable carrier, such as the diluents phosphatebuffered saline or a bicarbonate solution (e.g., 0.24 M NaHCO₃). Thecarriers used in the composition are selected on the basis of the modeand route of administration, and standard pharmaceutical practice.Suitable pharmaceutical carriers and diluents, as well as pharmaceuticalnecessities for their use, are described in Remington's PharmaceuticalSciences. An adjuvant, e.g., a cholera toxin, Escherichia coliheat-labile enterotoxin (LT), liposome, or immune-stimulating complex(ISCOM), can also be included in the new vaccine composition.

Skilled artisans can obtain further guidance in the preparation of aneurotoxin vaccine in Singh et al. (Toxicon., 27:403-410, 1990).Briefly, approximately 0.1 to 2 mg of neurotoxin (e.g., botulinum type Aneurotoxin or tetanus toxin, or attenuated variants thereof) and 0.1 to2 mg of biologically active Hn-33 (e.g., the Hn-33:neurotoxin ratio, byweight, can be 1:1 to 20:1) are added to a physiological buffer (e.g.,buffers at pH 4-7.5), and are incubated (e.g., at 4° C. to 30° C.) for atime sufficient to allow the Hn-33 to bind to the neurotoxin (e.g., 5minutes to 2 hours).

B. Therapeutic Compositions

Any disease or discomfort associated with an exaggerated release ofacetylcholine from a presynaptic nerve terminal can be treated with aHn-33 neurotoxin complex. These diseases are associated with eithersmooth or skeletal muscle spasms, such as spasmodic torticollis,essential tremor, spasmodic dysphonia, charley horse, strabismus,blepharospasm, oromandibular dystonia, spasms of the sphincters of thecardiovascular, gastrointestinal, or urinary systems, and tardivedyskinesia, which may result from treatment with an anti-psychotic drugsuch as THORAZINE® or HALDOL®.

To formulate the therapeutic compositions, Hn-33 is mixed with aneurotoxin in at least a 1:1, by weight, Hn-33:neurotoxin ratio in aphysiological buffer (e.g., phosphate buffered saline) at a suitabletemperature (0°-37° C.). The resulting Hn-33/neurotoxin complex can be,for example, lyophilized and resuspended in sterile, deionized water.Appropriate pharmaceutical carriers (e.g., the ones described above) canthen be added.

The therapeutic compositions can be formulated as a solution,suspension, suppository, tablet, granules, powder, capsules, ointment,or cream. In the preparation of these compositions, at least onepharmaceutical carrier is to be included. Examples of pharmaceuticalcarriers include solvent (e.g., water or physiological saline),solubilizing agent (e.g., ethanol, polysorbates, or Cremophor EL®),agent for making isotonicity, preservative, antioxidizing agent,excipient (e.g., lactose, starch, crystalline cellulose, mannitol,maltose, calcium hydrogen phosphate, light silicic acid anhydride, orcalcium carbonate), binder (e.g., starch, polyvinylpyrrolidone,hydroxypropyl cellulose, ethyl cellulose, carboxy methyl cellulose, orgum arabic), lubricant (e.g., magnesium stearate, talc, or hardenedoils), or stabilizer (e.g., lactose, mannitol, maltose, polysorbates,macrogols, or polyoxyethylene hardened castor oils) can be added. Ifnecessary, glycerin, dimethylacetamide, 70% sodium lactate, asurfactant, or a basic substance such as sodium hydroxide,ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine,or trisaminomethane is added. Biodegradable polymers such aspoly-D,L-lactide-co-glycolide or polyglycolide can be used as a bulkmatrix if slow release of the composition is desired (see e.g., U.S.Pat. Nos. 5,417,986, 4,675,381, and 4,450,150). Pharmaceuticalpreparations such as solutions, tablets, granules or capsules can beformed with these components. If the composition is administered orally,flavorings and colors can be added.

IV. Administration of Pharmaceuticals Compositions ContainingBiologically Active Hn-33

The new vaccine and therapeutic compositions can be administered via anyappropriate route, e.g., intravenously, intraarterially, topically byinjection, intraperitoneally, intrapleurally, orally, subcutaneously,intramuscularly, sublingually, intraepidermally, or rectally.

A. Vaccines

The amount of vaccine administered will depend, for example, on theparticular vaccine antigen, whether an adjuvant is co-administered withthe antigen, the type of adjuvant co-administered, the mode andfrequency of administration, and the desired effect (e.g., protection ortreatment), as can be determined by one skilled in the art. In general,the new vaccine antigens are administered in amounts ranging between 1μg and 100 mg per adult human dose. If adjuvants are administered withthe vaccines, amounts ranging between 1 ng and 1 mg per adult human dosecan generally be used. Administration is repeated as necessary, as canbe determined by one skilled in the art. For example, a priming dose canbe followed by three booster doses at weekly intervals. A booster shotcan be given at 8 to 12 weeks after the first immunization, and a secondbooster can be given at 16 to 20 weeks, using the same formulation. Seraor T-cells can be taken from the individual for testing the immuneresponse elicited by the vaccine against the neurotoxin. Methods ofassaying antibodies or cytotoxic T-cells against a specific antigen arewell known in the art. Additional boosters can be given as needed. Byvarying the amount of Hn-33, neurotoxin, or pharmaceutical composition,the immunization protocol can be optimized for eliciting a maximalimmune response.

Before administering the above compositions in humans, toxicity andefficacy testing in animals are desirable. In an example of efficacytesting, mice (e.g., Swiss-Webster mice) can be vaccinated via an oralor parenteral route with a composition containing Hn-33 and a neurotoxin(e.g., a tetanus toxin or type A botulinum neurotoxin). After theinitial vaccination or after optional booster vaccinations, the mice(and corresponding control mice receiving mock vaccinations) arechallenged with a LD₉₅ dose of the toxin. Efficacy is determined if micereceiving the Hn-33/toxin die at a rate lower than the mock-vaccinatedmice. Preferably, the difference in death rates should be statisticallysignificant. Rabbits can be used in the above testing procedure insteadof mice.

Alternatively, the new Hn-33/neurotoxin vaccine compositions can beadministered as ISCOMs. Protective immunity has been generated in avariety of experimental models of infection, including toxoplasmosis andEpstein-Barr virus-induced tumors, using ISCOMS as the delivery vehiclefor antigens (Mowat et al., Immunology Today, 12:383-385, 1991). Dosesof antigen as low as 1 μg encapsulated in ISCOMs have been found toproduce class I mediated cytotoxic T cell responses (Takahashi et al.,Nature, 344:873-875, 1990). Polypeptides are delivered into cells usingISCOMS in a manner and dosage similar to that described above forliposomes.

B. Therapeutic Compositions

The dose of the Hn-33/neurotoxin complex administered to a patient willdepend generally upon the severity of the condition, the age, weight,sex, and general health of the patient, and the potency of the toxin,which is expressed as a multiple of the LD₅₀ value for the mouse.

The dosages used in human therapeutic applications are roughlyproportional to the mass of muscle in need of treatment. Typically, thedose administered to the patient may be from about 0.01 to about 1,000units, for example, about 5 to 50 or 100, or 200 to 500 units, dependingon the particular use. A unit is defined as the amount ofHn-33/neurotoxin that kills 50% of a group of Swiss-Webster mice(typically a group of 18-20 female mice that weigh on average 20 grams).The dosage is adjusted, either in quantity or frequency, to achievesufficient reduction in acetylcholine neurotransmitter release to affordrelief from the symptoms of the disease or condition being treated.

Physicians, pharmacologists, and other skilled artisans are able todetermine the most therapeutically effective treatment regimen, whichwill vary from patient to patient. The potency of botulinum toxin andits duration of action means that doses are administered on aninfrequent basis (e.g., one 10-200 unit intramuscular injection, forexample, once every four to six months, or once every several weeks) andpreferably administered in an implant made from a polymer that allowsslow release of the toxin. Skilled artisans are also aware that thetreatment regimen must be commensurate with questions of safety and theeffects produced by the toxin.

Typically, the Hn-33/neurotoxin complex is suspended in aphysiologically acceptable solution, such as normal saline, and isadministered by an intramuscular injection. Prior to injection, carefulconsideration is given to the anatomy of the muscle group, in an attemptto inject the toxin complex into the area with the highest concentrationof neuromuscular junctions. If the muscle mass is not very great, theinjection can be performed with extremely fine, hollow, teflon-coatedneedles and guided by electromyography. The position of the needle inthe muscle should be confirmed prior to injection of the toxin, andgeneral anesthesia, local anesthesia, or other sedation may be used atthe discretion of the attending physician, according to the age andparticular needs of a given patient and the number of sites to beinjected.

As an example, an adult male patient suffering from tardive dyskinesiaresulting from treatment with an antipsychotic drug can be treated with50-200 units (as defined herein) of Hn-33/botulinum neurotoxin complexby direct injection into the facial muscles. Within three days, thesymptoms of tardive dyskinesia, i.e., orofacial dyskinesia, athetosis,dystonia, chorea, tics, and facial grimacing are markedly reduced.

Spasticity that occurs secondary to brain ischemia, or traumatic injuryof the brain or spinal cord, are similarly amenable to treatment.

In instances where the postsynaptic target is a gland, nerve plexus, organglion, rather than a muscle, the Hn-33/neurotoxin complex can beadministered to control profuse sweating, lacrimation, and mucoussecretion. For example, an adult male patient with excessive unilateralsweating can be treated by administering 0.01 to 50 units (as definedherein) of Hn-33/neurotoxin complex to the gland nerve plexus, ganglion,spinal cord, or central nervous system. Preferably, the nerve plexus organglion that malfunctions to produce the excessive sweating is treateddirectly. Administration of the Hn-33/neurotoxin complex to the spinalcord or brain, while feasible, may cause general weakness.

Other conditions that can be treated include tension headache and paincaused by sporting injuries or arthritic contractions. If necessary,overactive muscles can be identified with electromyography (EMG).

EXAMPLES

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

Example 1 Isolation of Biologically Active Hn-33

C. botulinum, Hall strain (obtainable as ATCC No. 3502) was grown in N-Zamine-based growth medium, and the Hn-33 isolated according to theprocedures described by DasGupta et al., Toxicon., 22:415-424 (1984) andmodified as follows. 32 liters of culture were grown in four-9L Pyrexcarboys at 37° C. without agitation. Cells were harvested after 10 daysof growth. After acid precipitation and centrifugation, the cell pelletswere extracted and digested with RNAse A. The digested crude extractswere then passed through two DEAE-Sephadex A-50 columns (2.5×60 cm) with0.05 M citrate buffer, pH 5.5. The column yielded a major elution peakin the void volume (FIG. 1). The first peak containing the crudebotulinum neurotoxin complex was pooled from the two columns andprecipitated with 0.351 g/ml ammonium sulfate solution.

The precipitated proteins were redissolved in 50 mM citrate buffer, pH5.5, and extensively dialyzed against the same buffer before beingloaded onto a G-100 Sephadex column (2.5×70 cm). Proteins were elutedwith the same buffer at a flow rate of 40 ml/hr. The furtherchromatographic separation of proteins resulted in two peaks (FIG. 2).Both the first and second peaks of the G-100 column elution werecollected for further analysis.

To isolate the type A botulinum neurotoxin, the crude neurotoxin complexwas applied to a second DEAE-A-50 column equilibrated as above andeluted with a 0-0.5 M NaCl gradient buffer. The neurotoxin in the firstelution peak was further purified by applying to a SP-C-50 column in 20mM sodium phosphate buffer (pH 7.9) and eluted with a 0-0.5 M NaClgradient buffer. The neurotoxin is contained in the second of twoelution peaks.

Electrophoretic analysis of the type A botulinum complex was carried outon the PhastSystem electrophoresis unit (Pharmacia LKB, Piscataway,N.J.) using a 4-15% gradient acrylamide gel. Electrophoretic data wereanalyzed with a CCD solid-state video camera (ITTI Inc. St. Petersburg,Fla.) for the molecular weight determination and for the estimation ofthe relative amount of each band. High molecular weight standards werepurchased from Bio-Rad and used for the determination of sample proteinmolecular weights.

The group of proteins obtained in the first elution band (FIG. 2) hasbeen referred to as the type A botulinum neurotoxin complex. The secondelution peak of the Sephadex G-100 column contained a single protein of33 kDa, otherwise called hemagglutinin-33 or Hn-33.

Estimation of the relative amounts of each component of the type Aneurotoxin complex was investigated via densitometry of the SDS-PAGEbands resulting from the first DEAE-A-50 eluted proteins. Undernonreducing conditions, the relative content of the band at 142 kDa was25%, whereas the band at 120 kDa (corresponding to the neurotoxin) was7%. Other components of neurotoxin associated proteins (NAPs) werepresent in significant proportions (54 kDa, 16%; 33 kDa, 25%; 20 kDa,9%; 17 kDa, 6%; and 14 kDa, 12%). Typically, the yield of purified Hn-33was about 2% after the Sephadex G-100 chromatography step.

Concentrations of complex (1.66 mg/ml) and neurotoxin (1.63 mg/ml) weredetermined according to the extinction coefficients ε (278 nm). Theconcentration of Hn-33 was determined by the method described inWhitaker et al., Anal. Biochem., 109:156-159 (1980), using the factor(A₂₃₅-A₂₇₈)/2.51. The number 2.51 in the denominator was empiricallydetermined to give the best correlation between absorbance and proteinconcentration and corrects for the contribution of absorbance byaromatic amino acids at 235 nm.

The molecular weight of Hn-33 was further confirmed by subjecting theisolated protein to matrix-assisted laser desorption mass spectrometry.15 pmoles of Hn-33 in sinapinic acid was analyzed on a Voyager-RPBiospectrometry Workstation (PerSeptive Biosystem, Boston, Mass.)according to manufacturer's instructions. The spectrometer was operatedat an accelerating potential of 30,000 V, and the protein was desorbedby a 336 W intensity laser. The spectrums were obtained with an averageof 174 scans. BSA was applied as a standard for assignment of molecularweights, using either singly or doubly charged ions.

The resulting mass spectrum (FIGS. 3A and 3B) indicated a major band at33,389 and significant peaks at 66,945 (dimer) and 100,359 (trimer). Aweak peak was observed at 133,717 (tetramer) as shown in the expandedgraph of FIG. 3B. Masses below 33,389 probably represent higher chargedspecies of Hn-33 or minor impurities.

The quaternary structure of isolated, biologically active Hn-33 wasfurther investigated using gel filtration chromatography. Hn-33 at 3.4mg/ml in 50 mM citrate buffer, pH 5.5, was applied to a Sephadex G-200column (1.5×80 cm). Standard elution volumes were obtained withcytochrome C, 12.4 kDa; carbonic anhydrase, 29 kDa; BSA, 66 kDa; alcoholdehydrogenase, 150 kDa; and apoferritin, 443 kDa. A plot of the log ofstandard MW values vs. standard Rf values (FIG. 4) and comparison withthe elution volume of Hn-33 indicated a molecular weight of 69 kDa whichis likely to be an Hn-33 dimer. In FIG. 4, the gray shading indicatesthe elution profile for standard MW proteins, and the black shadingindicates elution profile for Hn-33. This assay suggests that a trimericstructure predominates.

Example 2 Measuring Hemagglutination Activity of Isolated Hn-33

Biological activity was determined by the hemagglutination assay asdescribed in DasGupta et al. (1977), supra. Freshly drawn human blood(type A positive) was immediately treated with solution (GammaBiologicals, Inc., Houston, Tex.) and washed with 0.8% NaCl before use.Washed human erythrocytes (0.05 ml of 0.5% w/w suspension) were added to0.05 ml of type A botulinum toxin, Hn-33, or the neurotoxin complex in75 mM NaCl in wells of a U-Bottom microtiter plate (Falcon, BectonDickinson, N.J.). A twofold serial dilution, starting at a concentrationof 13.33 μg/ml of neurotoxin, Hn-33, or complex, was used for the assay.Hemagglutination activity was observed when the erythrocytes did notform into an annular shape or a dot in the bottom of the well after 4hours of incubation.

The purified Hn-33 was effective in eliciting hemagglutination in theabove assay at a concentration of 416 ng/ml, which was abolished in thepresence of 0.05% SDS. The hemagglutinating activity of the wholeneurotoxin complex (including all neurotoxin associated proteins) wasobserved at a minimum concentration of 52 ng/ml. The purified neurotoxindid not exhibit any hemagglutinating activity at concentrations of 13.33μg/ml or less.

To determine whether carbohydrates block the hemagglutination activityof Hn-33, 0.5 μg of isolated Hn-33 was mixed with serial dilutions ofo-nitrophenyl-β-D-galactoside or isopropyl-β-D-thiogalactoside, twosugars found on the apical surface of GI tract epithelial cells(starting with a 66.7 mM initial concentration), and then subjected tothe hemagglutination assay. At a 33 mM carbohydrate concentration, bothof the sugars mentioned above inhibited the Hn-33 hemagglutinationactivity, indicating that Hn-33 binds to these sugars. These resultssuggest that the new Hn-33/neurotoxin vaccine compositions bind to thesesugars on the surface of epithelial cells in the GI tract, facilitatingentry of Hn-33/neurotoxin vaccine compositions into the body.

In an attempt to investigate the contribution of different components ofthe neurotoxin complex to hemagglutination activity, Hn-33-specificantibodies were used to block hemagglutination. Hn-33-specificantibodies were isolated by fractioning a horse anti-type A neurotoxincomplex antiserum on a protein-G matrix column as described in Ogert etal., Anal. Biochem., 205:306-312 (1992), followed by fractionation on aHn-33 affinity column produced by attaching Hn-33 to Affigel 15 matrix(Bio-Rad, Hercules, Calif.). After applying the IgG fraction on theAffigel™ column, the column was washed with 0.15 M sodium phosphatebuffer, pH 7.0, and eluted with 0.2 M glycine.HCl.

The specificity of the Hn-33 antibodies was confirmed by a Western blotof type A neurotoxin complex proteins. The neurotoxin complex wasresolved on a 12.5% SDS-polyacrylamide gel (0.75 mm thick, 8×9 cmminigel) using a Mini-PROTEAN II® electrophoresis cell (Bio-Rad) underreducing conditions (2-mercaptoethanol added). Protein bands were thentransferred to a nitrocellulose membrane by transblotting them using aMini Trans-Blot electrophoretic transfer cell (Bio-Rad) according to theprocedure described in Towbin et al., Proc. Natl. Acad. Sci. USA,76:4350 (1979). The nitrocellulose membrane was treated with 3% BSAsolution in PBS for 1 hour at room temperature to block any non-specificbinding to antibodies in subsequent steps. The electroblotted proteinswere probed with the Hn-33 antibodies by suspending the membraneovernight at 4° C. in 50 μg/ml antibody solution dissolved in 3% BSA.Unbound antibodies were removed by washing the membrane with 0.05%Tween-20™ in PBS four times using 200 ml of solution for each washing.Subsequently, the membrane was incubated for 1 hour at room temperaturewith anti-horse IgG-peroxidase conjugate at a 1:10,000 dilution in 3%BSA solution. The bound antibodies were detected by chromogenicoxidation of α-chloronaphthol. This procedure indicated that theaffinity-purified Hn-33 antibodies recognized only a 33 kDa band.

Hn-33-specific antibodies completely blocked hemagglutination activityof Hn-33 and the complex at the higher antibody concentration.Hemagglutination activity elicited by 1.33 μg/ml of Hn-33 was blocked by412 ng/ml of Hn-33 antibody, but not 206 ng/ml of antibody. However, thehemagglutination activity elicited by 1.33 μg/ml of type A neurotoxincomplex was blocked by Hn-33 antibodies at both the 412 ng/ml and 206ng/ml concentrations. The latter result indicated that Hn-33 is the onlymajor hemagglutinin protein in the type A complex.

Example 3 Isolated, Biologically Active Hn-33 is Post-TranslationallyModified

The N-terminal amino acid sequence of isolated, biologically activeHn-33 was determined as follows. About 10 pmoles of purified Hn-33 wasdissolved in sample buffer (0.5 M sucrose, 15% SDS, 312.5 mM Tris, 10 mMEDTA) and subjected to electrophoresis on a 12.5% SDS-PAGE gel (0.75 mmthick, 8×9 cm mini-gel) using Mini-PROTEAN II electrophoresis cell(Bio-Rad). The proteins were then transferred to a PVDF membrane(Bio-Rad) in Towbin buffer (25 mM Tris, 192 mM glycine, and 20%methanol) using a Mini Trans-Blot electrophoretic transfer cell(Bio-Rad) at 60V overnight in an ice bath. Post-translational cleavagewas examined using Edman degradation followed by analysis on anautosequencer (Applied Biosystem Model 473A, Biomolecular ProteinSequencing Facility of Baylor College of Medicine, Houston, Tex.).

The same protein that had appeared as a single homogeneous band on aSDS-PAGE gel using 0.2 M tricine, 10 mM Tris-HCl, 1 mM EDTA, and 2.5%SDS (pH 8.0), appeared as three distinct bands on the gels used toprepare the protein for sequencing. To confirm the origin of thesebands, we sequenced the lower two bands, the upper band being identicalto the previously isolated Hn-33. Proteins in both bands had theidentical N-terminal sequence VIQNSLNDKI (SEQ ID NO:2). This sequencematched the sequence of Hn-33 at the +6 position (MEHYSVIQNSLNKDI; SEQID NO:3) as described in East et al., Syst. Appl. Microbiol., 17:306-312(1994). Therefore, the above result suggests that the mature proteinexists as a post-translational cleavage product.

Example 4 The Secondary Structure of Hn-33 Includes β-Sheets

To analyze the secondary structure of isolated, biologically activeHn-33, the protein was subjected to a far-UV circular dichroism (CD)study and FT-IR spectroscopy. CD spectra were recorded on a Jasco J715spectropolarimeter with a scanning speed of 20 nm per minute and aresponse time of 8 seconds, using a 1 mm pathlength quartz cuvette atroom temperature. The protein concentration used for spectral recordingswas 0.2 mg/ml. For the final comparison of control and SDS-treatedspectra, a reference spectrum of SDS solution was subtracted. Secondarystructure content was estimated by the method of Yang et al. (Meth.Enzymol., 130:208-269, 1986).

Since treatment of Hn-33 with 0.05% SDS abolished biological activity(see Example 2), CD spectra of Hn-33 were collected in the presence andabsence of submicellar (0.05%) and micellar (0.5%) concentration of SDSFIG. 5 is a series of spectra in which the solid line represents Hn-33without SDS, the wide dashed line represents Hn-33 in the presence of0.05% SDS, and the narrow dashed line represents Hn-33 in the presenceof 0.5% SDS. The CD spectrum indicated a major negative peak at 205 nmand a positive band at 187 nm. Treatment of Hn-33 with 0.05% SDSsignificantly changed the shape of the CD spectrum, showing a shift ofthe negative peak at 205 nm. The positive CD band shifted from 187 to190 nm. Treatment of Hn-33 with 0.5% SDS slightly increased the peak at205 nm as compared to the 0.05% SDS spectrum. Secondary estimationsuggested that Hn-33 was 6% α-helix, 55% β-sheets, and 39% random coils.In the presence of 0.05% SDS, the estimated composition shifted to 10%α-helix, 48% β-sheets, and 42% random coils. In the presence of 0.5%SDS, as compared to 0.05% SDS, the composition changed only in thepercentage of random coils, and then only very slightly, to 10% α-helix,48% β-sheets, and 41% random coils. Thus, treatment of Hn-33 with SDSincreased the α-helix content and decreased the β-sheet content of theHn-33 structure. However, little structural differences were seen in theHn-33 polypeptide under submicellar and micellar concentrations of SDS.

The secondary structure of Hn-33 was additionally analyzed using aNicolet 8210 FT-IR spectrometer as described in Fu et al., Appl.Spectrosc., 48:1432-1441 (1994). One ml of a solution containing 0.65mg/ml of Hn-33 was applied to a 60° horizontal zinc selenide,attenuated, total reflectance crystal. Spectra were built from 256 scansat a resolution of four per cm. All spectra were smoothed using anine-point Savitsky-Golay algorithm and were multiplied by a factor of100 for data analysis. An average spectrum was obtained from eightspectra which were recorded separately. For the secondary structureanalysis, data processing was modified from the method of Fu et al(1994), supra, by the use of LC software (LabCalc, Galactic IndustriesCo., Salem, N.H.). The Bassel deconvolution function was chosen with 3.3as the gamma factor and 0.355 as the filter factor. The deconvolvedspectra were first fitted with fixed band positions and floatedhalf-height band width, and the band shaped to obtain the mostreasonable band intensity for all individually resolved bands. Fixedband positions were released to obtain the best curve-fitting results. Acomputer calculation was used to float each parameter until the best fit(as determined by the best root mean square value) was obtained. Theresulting parameter set was then applied to fit the bands into theoriginal protein spectra to acquire the best resolved underlying bands.The amide I region was analyzed from 1550 to 1750 cm⁻¹, and the amideIII region was analyzed from 1400 to 1200 cm⁻¹ to eliminate thecontribution from the neighboring bands. The band assignments forvarious secondary structures were based on assignments used in Fu et al.(1994), supra.

The amide I and III IR spectra of Hn-33 are shown in FIGS. 6A and 6B.Major absorption bands were observed at 1632 and 1237 cm⁻¹ in amide Iand amide III regions, respectively, suggesting that β-sheets dominatethe Hn-33 structure. Resolved band positions of amide I and III andtheir contributions and assignments to secondary structure types weredetermined. Based on the amide I band analysis, Hn-33 consists of 74%β-sheets, no α-helix, and 26% β-turns or unordered structures. Based onthe amide III band analysis, Hn-33 consists of 77% β-sheets, no α-helix,and 23% β-turns or unordered structures. Despite small differences inthe individual secondary structure types, data from the two amideregions were generally consistent and agreed with the far-UV CD analysisabove.

The broad band observed at 1370 cm⁻¹ (FIG. 6B) is not commonlyassociated with proteins and may be indicative of a prosthetic group. Weused a Schiff's reagent kit (Sigma, St. Louis, Mo.) per manufacturer'sinstructions and as described in Doerner et al., Anal. Biochem.,187:147-150 (1990) to test for carbohydrates. No carbohydrates werefound.

Example 5 Isolated, Biologically Active Hn-33 is Protease Resistant

Biologically, active Hn-33 was isolated as described in Example 1 above.Digestion of Hn-33 was carried out as described in Deresiewicz et al.,Biochemistry, 33:12844-12851 (1994). The degree of proteolyticfragmentation was monitored by performing SDS-PAGE on the digestionreaction followed by Western blotting as described above. Results aretypically expressed as a percentage of intact polypeptide, referring tothe amount of the polypeptide remaining as a full-length band on theSDS-PAGE gel. The percentage degraded is one hundred minus thepercentage intact.

A relatively high concentration of trypsin (up to 1 mg/ml for 24 hours)did not degrade Hn-33. Under milder conditions (0.1 mg/ml for 30minutes), the type A neurotoxin complex was degraded. The Hn-33 wasresistant to proteolytic attack from all proteases tested, includingα-chymotrypsin, subtilisin, and pepsin. Resistance was also observedunder conditions of low pH (see conditions for pepsin below) or addedcarbohydrates.

The trypsin digestions was carried out by incubating Hn-33 with theprotease at a 250:1 w/w ratio in 50 mM Tris (pH 7.0) and 5 mM CaCl₂.When carbohydrates (o-nitropheny-β-D-galactoside andisopropyl-β-D-thiogalactoside) were optionally added to a finalconcentration of 66.7 mM (a concentration that inhibited Hn-33hemagglutination activity), the incubations were carried out at 25° C.for 30 minutes.

Digestions with other proteases were as follows: Pepsin digestions werecarried out at a 25:1 protein: protease ratio in 50 mM ammonium acetate(pH 2.0) and 2 mM EDTA. α-Chymotrypsin digestions were carried out at a1000:1 protein:protease ratio in 50 mM Tris (pH 7.8) and 5 mM CaCl₂.Subtilisin digestions were carried out at a 25:1 protein:protease ratioin 0.1 mM ammonium bicarbonate (pH 7.5) and 1 mM guanidine-HCl. Trypsinand α-chymotrypsin digestions were carried out at room temperature, andthe pepsin and subtilisin digestions were carried out at 37° C.Digestions were stopped by the addition of 1 mM PMSF into the reactions.Stopped reactions were then boiled in SDS-PAGE running buffer beforeloading onto 8-25% gradient gels. Hn-33 was highly resistant (less than5% of the polypeptide digested as shown on SDS-PAGE gels) to allproteases tested herein.

These results indicate that Hn-33 is resistant to a variety of proteasesfound in the GI tract and suggests that the polypeptide (or fragments orderivatives thereof) is stable if administered to the GI tract and wouldthus be useful to stabilize an oral vaccine composition.

Example 6 Isolated, Biologically Active Hn-33 Binds to Type A Neurotoxinand Protects the Resulting Complex from Proteolytic Degradation

Type A neurotoxin and Hn-33 were purified from C. botulinum type A asdescribed in Example 1 above.

To determine direct binding between the neurotoxin and Hn-33, 5 mg ofthe neurotoxin was mixed with 1.7 mg Hn-33 in 3 ml 0.05 M sodiumcitrate, pH 5.5 (this corresponds to a 1:1.5 neurotoxin to Hn-33 molarratio). The mixture was incubated for 30 minutes at room temperaturebefore being applied to a Sephadex™ G-200 column (1.5×100 cm) which waspreviously equilibrated with 0.05 M citrate buffer, pH 5.5. The proteinwas eluted with the same buffer at a flow rate of 12 ml/h. The 1.7 mlfractions were collected, and the protein content was estimated bymonitoring absorbance at 280 nm. Peak fractions were analyzed on 8-25%SDS-PAGE gels.

Two mg each of standard proteins (cytochrome C, 12.4 kDa; carbonicanhydrase, 29 kDa; BSA, 66 kDa; alcohol dehydrogenase, 150 kDa; andapoferritin, 443 kDa) were dissolved in 0.05 M sodium citrate, pH 5.5,and applied to Sephadex G-200 column and eluted in the manner describedimmediately above. Blue dextran was run along with the standard proteinsto estimate the void volume of the column. Molecular weights of thepeaks corresponding to Hn-33 and its complex with neurotoxin wereestimated using a plot of the log of standard MW vs. standard Rf value.

The Hn-33/neurotoxin mixture was eluted in two peaks (FIG. 7)0. Peakfractions were subjected to SDS-PAGE as described in Example 1, followedby Coomassie Blue staining, indicating that the first peak contained a140 kDa and a 33 kDa protein and the second peak contained only the 33kDa protein. Based on the 280 nm absorbance and the SDS-PAGE, it wasestimated that about 94% of the Hn-33 bound to the neurotoxin.

Neurotoxin alone, neurotoxin complex, or equal amounts of neurotoxin andHn-33 (which were mixed together and incubated for 30 minutes at roomtemperature), were dialyzed for 30 minutes in digestion buffer specificfor each protease and then subjected to digestions as described inExample 2. As expected, the neurotoxin complex remained at least 60%intact in digestions containing pepsin, trypsin, subtilisin, andα-chymotrypsin.

Pepsin began to digest the “naked” neurotoxin after 20 minutes andcompletely digested the neurotoxin after 60 minutes. When Hn-33 wasincluded in the “naked” neurotoxin incubation, the neurotoxin remainedat least 75% intact even after 90 minutes of digestion. Similar resultswere obtained when trypsin and chymotrypsin were used in separateexperiments. These experiments indicate that Hn-33 can protectneurotoxins from proteolytic degradation under conditions which mimicthe GI environment, including low pH and the presence epithelial cellsurface carbohydrates. Thus, isolated, biologically active Hn-33 shouldprotect a botulinum neurotoxin protein from proteolytic degradation.

Example 7 Use of Isolated, Biologically Active Hn-33 in Vaccines

One milligram of isolated, biologically active Hn-33 is incubated withone milligram of type A botulinum neurotoxin in phosphate-bufferedsaline to prepare an Hn-33 vaccine composition. After an incubationsufficient to allow binding of Hn-33 to the neurotoxin, the mixture islyophilized and resuspended in 100 milliliters of sterile, deionizedwater to formulate the vaccine. The vaccine is administered by drinking.

For testing of the vaccine in an animal model, a rabbit is orallyimmunized via a feeding needle inserted into the rabbit's stomach with avaccine containing 200 micrograms of the Hn-33/neurotoxin complexprepared as described above in this Example. The same quantity of salinewithout neurotoxin is administered to additional rabbits of the sametype and weight as a control. Such vaccine administration is describedin Karem et al., Infect. Immun., 63:4557-4563 (1995). Rabbits areobserved for signs of botulism symptoms. Two booster injections are madeat a dosage of 200 micrograms Hn-33/neurotoxin complex each, the firstbooster at two weeks post-initial inoculation and the second booster atsix weeks post-initial inoculation. Rabbits are again observed for signsof botulism.

After the initial and booster immunizations, sera and T-cells are testedfor an immune response against the neurotoxin vaccine. All rabbits arethen subcutaneously challenged with an LD₅₀ does of the correspondingneurotoxin or neurotoxin complex. Any changes in symptoms and deathrates in immunized rabbits as compared to controls are noted andanalyzed.

Example 8 Isolated, Biologically Active Hn-33 Enhances BotulinumNeurotoxin Protease Activity

To determine if Hn-33 could enhance the protease activity of the type Abotulinum neurotoxin protein, the ability of the neurotoxin to cleave aSNAP-25-GST fusion protein in the presence and absence of Hn-33 wasexamined.

SNAP-25 is a membrane-associated protein implicated in the fusion ofsynaptic vesicles with the presynaptic membrane. Botulinum neurotoxincleaves SNAP-25 at a site 9 and 26 amino acids from the C-terminus(Williams et al., J. Biol. Chem., 271:7694-7699, 1996; and Sollner etal., Nature, 362:318-324, 1993).

The SNAP-25-GST fusion protein was purified using standard methods whichare described in Smith et al., Meth. Molec. Cell. Biol., 4:220-229(1993). The purified fusion protein was dissolved and dialyzed in 50 mMTris, 10 mM sodium phosphate, 300 mM NaCl, 2 mM MgCl₂, 0.3 mM CaCl₂, 1mM 2-mercaptoethanol, and 0.1% NaN₃ (pH 7.6, 4° C.).

Type A botulinum neurotoxin was purified as described in Example 1above. Type E botulinum neurotoxin was purified as described in Gimenezet al., Appl. Environ. Microbiol., 53:2827-2830 (1987) and modified asfollows. The precipitated type E neurotoxin was dissolved in anddialyzed against 0.02 M sodium phosphate, pH 7.4, for 12 to 16 hours.The complex was then applied to a DEAE-Sephadex A-50 column equilibratedwith the buffer immediately above. Unbound material was washed off with10 column volumes of the buffer while most of the complex bound to thecolumn. The type E neurotoxin was eluted with 0.2 M NaCl gradient untilthe A280 readings reached background levels.

Digestions were carried out in 0.01 M phosphate buffer (pH 7.4)containing 1.7 μM neurotoxin and 5 μM SNAP-25 fusion protein for 30minutes at 37° C. 5 μg of Hn-33 was optionally added before thedigestion. The digestions were optionally pre-incubated with 20 mM DTTfor 30 minutes at 37° C. to reduce disulfide bonds. Digestions were thenstopped by adding SDS-PAGE sample buffer and heating the digestion for 4minutes at 100° C. A 30 μl sample was then resolved on a 12% SDS-PAGEgel and Western blotted using an antibody raised against the C-terminal12 amino acids of SNAP-25. The amount of substrate cleaved wasdetermined as described in Example 6 above.

Type A neurotoxin cleaved 57% of the SNAP-25 fusion protein under theconditions described above while type E neurotoxin cleaved only 28% ofthe SNAP-25 fusion protein. In the presence of Hn-33, the type Aneurotoxin cleaved 80% of the fusion protein and the type E neurotoxincleaved 47% of the fusion protein (FIG. 8). Thus, addition of Hn-33 tothe neurotoxins before digestion resulted in enhancement of substratecleavage by botulinum neurotoxins, indicating that a therapeuticcompositions containing Hn-33 and a neurotoxin can have a highertherapeutic activity. Such improved compositions are useful for patientsrequiring more neurotoxic activity or less administered drug due toside-effects caused by non-neurotoxin ingredients.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the claims. Other aspects,advantages, and modifications are within the scope of the followingclaims.

1-7. (canceled)
 8. A method of eliciting an immune response against aneurotoxin in an animal, the method comprising administering to theanimal an amount of a composition effective to elicit an immune responseagainst the neurotoxin, the composition comprising an isolated,biologically active Hn-33 of type A Clostridium botulinum and aneurotoxin.
 9. The method of claim 8, wherein the composition furthercomprises an adjuvant.
 10. The method of claim 8, wherein the neurotoxinis selected from the group consisting of type A botulinum neurotoxin,type E botulinum neurotoxin, and tetanus toxin.
 11. The method of claim8, wherein the neurotoxin is type A botulinum neurotoxin.
 12. The methodof claim 8, wherein the composition is administered orally to theanimal.
 13. The method of claim 8, wherein the composition furthercomprises a biodegradable polymer that enables slow release of theneurotoxin.
 14. The method of claim 8, wherein the animal is a human.15-23. (canceled)